Intensive research has been carried out into, for example, increasing the sensitivity and simplifying the measurement systems for the analysis and measurement of specific materials in various fields such as clinical diagnosis, food hygiene, and environmental hygiene. In particular, immunoassays employing an antigen-antibody reaction are more widely employed thereamong, for reasons of sensitivity and specificity. The sandwich enzyme immunoassay (EIA) method can be cited as a representative immunoassay method.
In sandwich EIA, of two types of antibody for a subject material to be measured, one is immobilized on a solid phase; this solid phase, the other antibody which is labeled with an enzyme, and a test sample are mixed together and reacted; after removing unreacted enzyme-labeled antibody by washing, an enzyme substrate solution is added and the amount of immunocomplex generated is measured by the enzyme activity. Since this method has good reproducibility and, furthermore, the antigen concentration can be measured with high sensitivity, it is widely employed. However, the sandwich method has the problem that a complicated and time-consuming washing operation for removing unreacted labeled antibody is necessary, etc.
In order to solve this problem, in recent years immunoassay methods employing energy transfer phenomena such as fluorescence energy transfer (FRET), bioluminescence energy transfer (BRET), and chemiluminescence energy transfer (CRET), or intermolecular interactions such as the complementation of enzyme activity of enzyme molecules of which a part has been deleted or altered have been noteworthy. Fluorescence energy transfer is a phenomenon observed between certain specific fluorescent materials; in a case where the fluorescence spectrum of a fluorescence energy donor and the excitation light spectrum of a fluorescence energy acceptor overlap and, moreover, where the distance between these two molecules approaches 10 nm or less, the fluorescence energy of the fluorescence energy donor transfers to the acceptor, and luminescence of the fluorescence energy acceptor is observed.
Bioluminescence energy transfer is a phenomenon observed between a fluorescent material and an enzyme, such as firefly luciferase, that catalyses bioluminescence; in a case where the luminescence spectrum of the bioluminescence enzyme and the excitation light spectrum of a luminescence energy acceptor overlap and, moreover, where the distance between these two molecules approaches 10 nm or less, the luminescence energy of the bioluminescence energy donor transfers to the acceptor, and fluorescence of the luminescence energy acceptor is observed.
Chemiluminescence energy transfer is a phenomenon observed between a fluorescent material and an enzyme, such as a peroxidase, that catalyses chemiluminescence; in a case where the luminescence spectrum of the chemiluminescence enzyme and the excitation light spectrum of a luminescence energy acceptor overlap and, moreover, where the distance between these two molecules approaches 10 nm or less, the luminescence energy of the chemiluminescence energy donor transfers to the acceptor, and fluorescence of the luminescence energy acceptor is observed.
The complementation of enzyme activity of enzyme molecules of which a part has been deleted or altered is a phenomenon typified by the β-galactosidase Δα mutant in which the α site has been deleted, and the Δω mutant in which the ω site has been deleted; by themselves Δα and Δω have reduced β-galactosidase enzyme activity, but when the two approach each other the β-galactosidase enzyme activity increases as a result of association. Several examples have been reported so far in which such energy transfer phenomena and such complementation of enzyme activity of enzyme molecules of which a part has been deleted or altered are applied to immunoassay and the detection of protein-protein interaction.
Among these, as an example of the application of the energy transfer phenomena to an immunoassay there can be cited a method reported by Toyobo Co., Ltd. (JP, A, 10-319017). In this method, materials A and B that have the capacity to bind to a subject material (X) to be measured are labeled with materials F1 and F2 respectively, which have a fluorescence energy donor-acceptor relationship; A-F1, X, and B-F2 are mixed and reacted for a fixed time, and after forming a complex F1-A:X:B-F2, without carrying out a washing operation, the amount of F1-A:X:B-F2 is measured as the efficiency of fluorescence energy transfer from F1 to F2.
However, the actual subject material to be immunoassayed (antigen) is often a high molecular weight protein and, furthermore, when two types of monoclonal antibody are used, etc., since the two epitopes are spatially separated, even if the reaction complex F1-A:X:B-F2 is formed, F1 and F2 in the F1-A:X:B-F2 complex are not in spatial proximity, and efficient energy transfer cannot take place. As a result, there is the problem that the sensitivity of the measurement is low.
According to Förster's theory of fluorescence energy transfer, the efficiency of fluorescence energy transfer is inversely proportional to the sixth power of the distance between the donor and acceptor (Anal. Biochem, 218, 1-13, 1994). Thus, when applying the FRET, BRET, CRET, etc. energy transfer phenomena to an immunoassay, it is necessary to establish a technique that always arranges the luminescence energy donor and acceptor in fixed positions within this type of immunocomplex.
In the application of FRET to immunoassay, the method of Ueda et al. (JP, A, 10-78436) can be cited as an example in which the spatial arrangement of the energy donor and acceptor is carried out well. In this method, antibody VL and VH fragments are labeled with fluorescence energy donor and acceptor and, in the presence of an antigen, fluorescence energy transfer takes place only when a stable three-way association of antigen, VL, and VH is formed, and by measuring the fluorescence intensity ratio of the fluorescence energy donor and acceptor, the antigen concentration is measured. It has been demonstrated (Anal. Biochem, 289, 77-81, 2001) that this measurement method employing VL and VH can measure the antigen concentration in BRET also.
This method, which combines, with fluorescence energy transfer, the property of VL and VH antibody fragments always being within 5 nm when an antigen is present, is an exceptionally unique immunoassay method, but since for many of the antibodies present in nature VL and VH have the property of associating even in the absence of an antigen, this principle cannot be applied to all antibodies. As a result, it cannot be put to practical use.
Furthermore, a method in which the complementation of enzyme activity of enzyme molecules of which a part has been deleted or altered was applied to monitoring protein-protein interaction has been reported by Rossi et al. (Proc. Natl. Acad. Sci. USA, 94, 8405-8410, 1997). In the method of Rossi et al., expression vectors of the proteins FRAP and FKBP12, for which dimerization is induced by rapamycin, fused with Δα and Δω β-galactosidases respectively were introduced into mammalian cells; when rapamycin was added to these chimeric protein-expressing cells, dimerization of the FRAP and FKBP12 proteins took place due to the rapamycin, and as a result rapamycin concentration-dependent β-galactosidase activity could be measured.
This method is an extremely good method for carrying out measurement of a material in viable cells, but it is an extremely special example in which the low molecular weight rapamycin-induced dimerization of FRAP and FKBP is utilized. When using this kind of enzyme complementation in the detection of an immunocomplex in immunoassay, in the same way as immunoassay methods employing energy transfer such as FRET, a technique that always arranges reporter proteins in fixed positions is also necessary.
Therefore, when intermolecular interactions such as an energy transfer phenomenon like fluorescence energy transfer (FRET) or luminescence-fluorescence energy transfer (BRET) and the complementation of enzyme activity of enzyme molecules of which a part has been deleted or altered are used in material concentration measurement detection systems, in order to carry out measurement with good precision and high sensitivity, the present invention provides a method for controlling the spatial arrangement of the energy donor and energy acceptor in the reaction complex, or the spatial arrangement of the complementation reaction donor and the complementation reaction acceptor, and a measurement system to which the method is applied.